We present a simple and efficient method for expressing cDNAs in Purkinje neurons (PNs) present in heterogeneous cerebellar cultures from mouse. The method combines the transfection of freshly-dissociated cerebellar cells via nucleofection with the use of novel expression plasmids containing a fragment of the L7 (Pcp-2) gene that, within the cerebellum, drives PN-specific expression. The PN transfection rate (determined 13 days post nucleofection) is approximately 70%, and double and triple transfections are feasible. Expression in PNs is obvious after one week in culture and still strong after three weeks, by which time these neurons are well-developed. Moreover, high-level expression is restricted almost exclusively to the PNs present in these mixed cultures, which greatly facilitates the characterization of PN-specific functions. As proof of principle, we used this method to visualize (1) the morphology of living PNs expressing mGFP, (2) the localization and dynamics of the dendritic spine proteins PSD-93 and Homer-3a tagged with mGFP and (3) the interaction of live PNs expressing mGFP with other cerebellar neurons expressing mCherry from a &#946;-actin promotor plasmid. Finally, we created a series of L7-plasmids containing different fluorescent protein cDNAs that are suited for the expression of cDNAs of interest as N- and C-terminally tagged fluorescent fusion proteins. In summary, this procedure allows for the highly efficient, long-term, and specific expression of multiple cDNAs in differentiated PNs, and provides a favorable alternative to two procedures (viral transduction, ballistic gene delivery) used previously to express genes in cultured PNs.